CN109536503B - 一种靶向结合cd133蛋白的核酸适配体及其筛选方法和用途 - Google Patents
一种靶向结合cd133蛋白的核酸适配体及其筛选方法和用途 Download PDFInfo
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Abstract
本发明提供了一种靶向结合CD133蛋白的核酸适配体及其筛选方法和用途，核酸适配体序列如SEQ ID NO.1，本发明采用细胞筛选的方法从ssDNA文库中筛选出靶向结合CD133蛋白的适配体，并对筛选出的ssDNA通过高通量测序的方法进行检测，最终通过流式细胞术等方法对所得序列的稳定性以及亲和性的分析，挑选出适配体AP‑1‑M。然后通过免疫荧光法证明适配体AP‑1‑M能够特异性地靶向结合CD133蛋白，并和表达CD133蛋白的ATC细胞结合从而起到靶向输送的作用，为ATC的靶向治疗提供新的思路和方法。
Description
技术领域
本发明属于生物制药领域，特别涉及一种靶向结合CD133蛋白的核酸适配体。
背景技术
甲状腺癌(thyroid carcinoma)是最常见的甲状腺恶性肿瘤，约占全身恶性肿瘤的1％。按照其恶性程度可分为分化型甲状腺癌(DTC)，低分化型甲状腺癌(PDTC)，未分化型甲状腺癌(ATC)。ATC由于能迅速扩展到颈部，会引起呼吸窘迫和食管梗阻，极易出现淋巴结和远处转移，其中位生存期少于6个月，甚至1年内的生存率只有10％。然而，目前人们对ATC的分子机制尚不明确，诊断和治疗方法有限，因此，迫切的需要更好的诊断和治疗的方法。
CD133蛋白为细胞膜蛋白超家族成员，含有865个氨基酸、分子量约为120kDa的糖蛋白，其分子结构包括：细胞外N-端，2个细胞外Loop环，5次跨膜结构域，一个59个氨基酸的细胞C-端和8个N-糖基化端。CD133蛋白的预测大小为97kDa，但实际的糖基化CD133的分子量为120kDa。近年来，研究者发现CD133蛋白存在于多种干细胞样的肿瘤细胞表面，其中包括肝癌、结肠癌以及卵巢癌。
核酸适配体，又称化学抗体，具有精准的靶向性，由单链的核糖核苷酸或脱氧核糖核苷酸构成。虽然多数核酸适配体本身没有治疗作用，但核酸适配体可以与毒性药物相连接组成靶向适配体偶联物，可以将药物“精确”地运送到靶细胞，使得适配体偶联药物被靶细胞内吞，在细胞内部释放药物诱导凋亡，既有效地提高了肿瘤局部的药物浓度，又可极大地降低体内其它组织、器官的毒性，从而达到真正靶向增效减毒的作用。
CD133蛋白存在于多种干细胞样的肿瘤细胞表面，其中包括肝癌、结肠癌以及卵巢癌，这为临床上筛选和研究新型药物提供了较好的特异性靶点，从而为研发更加安全有效的分子靶向药物提供了的思路。
此外，也有研究报道CD133蛋白在未分化型甲状腺癌(Anaplastic thyroidcarcinoma，ATC)中有表达，但是目前缺少合适的适配体用于未分化型甲状腺癌治疗研究。
发明内容
本发明的膜蛋白CD133为适配体，能够靶向结合。可以用于ATC的靶向治疗及研究。
本发明的目的是提供一种用于靶向结合CD133蛋白的单链DNA核酸适配体。
根据本发明的其中一方面，提供了一种靶向结合CD133蛋白的核酸适配体，该靶向结合CD133蛋白的核酸适配体为单链DNA核酸适配体。
根据本发明的其中一方面，提供了一种靶向结合CD133蛋白的核酸适配体，该靶向结合CD133蛋白的核酸适配体序列如序列表中SEQ ID NO.1
，其序列为：
5’-TACCAGCCGTTTCCCCGGAGGGTCACCCCTGACGCATTCGGTTGAC-3’。
根据本发明的其中一方面，提供了一种筛选靶向结合CD133蛋白的核酸适配体的方法，方法步骤为：
a、采用细胞筛选的方法从ssDNA文库中筛选出靶向结合CD133蛋白的适配体；
b、对步骤a筛选出的ssDNA通过高通量测序的方法进行检测，并对序列的稳定性以及亲和性的分析，筛选出靶向结合CD133蛋白的核酸适配体；
c、对步骤b筛选出的上述靶向结合CD133蛋白的核酸适配体通过免疫荧光法验证上述靶向结合CD133蛋白的适配体能够特异性地靶向结合CD133蛋白，并和表达CD133蛋白的ATC细胞结合。
根据本发明的其中一方面，提供了一种筛选靶向结合CD133蛋白的核酸适配体的方法，筛选方法筛选出的其中一种靶向结合CD133蛋白的核酸适配体的序列如序列表中SEQID NO.1即AP-1-M。
本发明的AP-1-M的5’端连接上8对GC碱基序列，AP-1-M可以直接携带细胞毒性药物(如阿霉素)，形成AP-1-M-Dox药物偶联物，选择性的杀伤CD133阳性的ATC细胞。因此，本发明AP-1-M-Dox在制备用于治疗CD133阳性的肿瘤(如ATC)制剂中的应用。
根据本发明的其中一方面，提供了一种靶向结合CD133蛋白的核酸适配体的应用，序列如序列表中SEQ ID NO.1的靶向结合CD133蛋白的核酸适配体(AP-1-M)在未分化型甲状腺癌治疗药物中的应用。
根据本发明的其中一方面，提供了一种靶向结合CD133蛋白的核酸适配体的应用，靶向结合CD133蛋白的核酸适配体(AP-1-M)偶联毒性药物构成未分化型甲状腺癌靶向治疗药物。
根据本发明的其中一方面，提供了靶向结合CD133蛋白的核酸适配体的应用，靶向结合CD133蛋白的核酸适配体偶联毒性药物构使用通过在上述靶向结合CD133蛋白的核酸适配体的5’端连接上8对GC碱基序列从而结合细胞毒性药物。
以经典化疗药物阿霉素耦联核酸适配体AP-1-M，得到AP-1-M-Dox并确证其确有较好的细胞毒性作用，而且由于核酸适配体AP-1-M可以选择性的结合CD133阳性的肿瘤细胞，而与CD133阴性的细胞结合较弱，因此本发明的又一方面，提供适配体作为CD133阳性的肿瘤化疗靶向药物载体的应用，将化疗药物结合于核酸适配体上，进行肿瘤放化疗时，可以选择性地杀伤CD133阳性的肿瘤细胞，而对正常细胞没有毒性，上述的化疗药物，可以是选自代谢类药物如甲氨蝶呤、氟尿嘧啶、氟尿苷、吉西他滨、雷替曲塞，抗癌抗生素类药物如丝裂霉素C、博来霉素、多柔比星、表柔比星、吡柔比星，植物碱类如长春碱、紫杉醇、羟基喜树碱，抗肿瘤激素类如他莫昔芬、来曲唑、强的松，杂类如顺铂、卡铂、米妥蒽醌，抗肿瘤小分子靶向药物如吉非替尼、伊马替尼或拉帕替尼，更优选地，上述化疗药物选自氟尿嘧啶、氟尿苷、甲氨蝶呤、顺铂、吉西他滨、雷替曲塞、丝裂霉素、博莱霉素、长春碱、紫杉醇、羟基喜树碱、卡铂、他莫昔芬、来曲唑、强的松、吉非替尼、伊马替尼或拉帕替尼。
根据本发明的其中一方面，提供了靶向结合CD133蛋白的核酸适配体的应用，细胞毒性药物为阿霉素。
本发明靶向结合CD133蛋白的核酸适配体的整体思路为：
采用细胞筛选的方法从ssDNA文库中筛选出靶向结合CD133蛋白的适配体，并对筛选出的ssDNA通过高通量测序的方法进行检测，最终通过流式细胞术等方法对所得序列的稳定性以及亲和性的分析，挑选出适配体。然后通过免疫荧光法证明适配体能够特异性地靶向结合CD133蛋白，并和表达CD133蛋白的ATC细胞结合从而起到靶向输送的作用。这为ATC的靶向治疗提供新的思路和方法。
本发明靶向结合CD133蛋白的核酸适配体还具有以下优势：1.为体外筛选，可快速人工合成；2.分子量不大，不发生免疫反应，易与靶分子特异性结合，亲和力强，且解离常数可以达到pmol至nmol；3.化学稳定性好，变性与复性可逆，易于室温运输和长期保存等。
附图说明
图1是本发明其中一实施例中不同细胞株中CD133蛋白的表达及定位情况图；
图2是本发明其中一实施例适配体AP-1-M的特异性检测图；
图3是本发明其中一实施例适配体AP-1-M和FRO细胞的结合情况检测。
图4是本发明其中一实施例适配体AP-1-M和Dox的偶联合成方式示意图；
图5是本发明其中一实施例适配体AP-1-M的特异性检测图。
具体实施方式
下面结合附图对本发明作进一步的说明。
实施例1：靶向结合CD133蛋白的核酸适配体的筛选
a、采用细胞筛选的方法从ssDNA文库中筛选出靶向结合CD133蛋白的适配体；
b、对步骤a筛选出的ssDNA通过高通量测序的方法进行检测，并对序列的稳定性以及亲和性的分析，筛选出靶向结合CD133蛋白的核酸适配体；
c、对步骤b筛选出的靶向结合CD133蛋白的核酸适配体通过免疫荧光法验证靶向结合CD133蛋白的适配体能够特异性地靶向结合CD133蛋白，并和表达CD133蛋白的ATC细胞结合。
筛选出的其中一种靶向结合CD133蛋白的核酸适配体的序列如序列表中SEQ IDNO.1即AP-1-M。
实施例2：不同细胞株中CD133蛋白的表达及定位情况
如图1所示，Caco-2：结肠腺癌细胞；FRO：未分化型甲状腺癌细胞；NTH：正常甲状腺细胞；293T：人胚肾细胞株。
利用共聚焦显微镜(Confocal Microscopy)观察不同细胞株中CD133蛋白的表达情况(图1)，红色荧光(即图1中左下角四张显微照片中膜状分布的亮点)代表膜蛋白CD133蛋白的表达分布，蓝色荧光(即图1各显微照片中核状亮斑)代表DAPI，指示细胞核。结果显示，未分化型甲状腺癌FRO细胞和作为阳性对照组的结肠腺癌细胞Caco-2细胞中CD133蛋白表达于细胞膜上，而作为阴性对照的HEK-293T细胞和正常甲状腺细胞Nthy-ori3-1(NTH)不表达CD133蛋白。
实施例3：适配体AP-1-M与CD133阳性细胞及阴性细胞结合。
如图2所示，通过利用FITC标记AP-1-M核酸适配体，以过表达CD133蛋白的HEK-293T细胞作为阳性细胞、野生型的HEK-293T细胞为阴性细胞，在4℃环境下将AP-1-M核酸适配体和阳性细胞及阴性细胞孵育，然后在激光共聚焦显微镜下观察发现AP-1-M和阳性细胞结合并定位于细胞膜上(图2-A)，而和阴性细胞不结合(图2-B)，这也说明了AP-1-M是针对CD133蛋白的靶向核酸适配体。
结果表示，AP-1-M是特异性结合CD133蛋白的核酸适配体，且用流式细胞术测核酸适配体的亲和性曲线得出，AP-1-M的Kd值为101.4nM。
实施例4：适配体CD133和未分化型甲状腺癌细胞FRO的结合。
如图3所示，将AP-1-M和FRO细胞分别在4℃和37℃环境中孵育30分钟，发现在4℃环境下时，由于细胞的内吞作用被抑制，AP-1-M和FRO细胞结合并位于细胞膜上(图3-A)；而在37℃环境下由于细胞的内吞作用，AP-1-M被FRO细胞内吞入胞内(图3-B)。
结果说明AP-1-M可以和FRO细胞结合且位于细胞膜上并可被内吞入胞内从而达到靶向输送的目的。
实施例5：适配体AP-1-M偶联药物的方法
如图4所示，本发明研究优选富含GC碱基对的DNA序列，由于富含GC碱基对的DNA序列可以自身发生聚合，形成口袋结构，从而物理镶嵌含有恵环的药物，如阿霉素，肉红霉素等，利用阿霉素有自身的荧光光谱，且在GC碱基对中被猝灭的特性，检测和计算Aptamer的载药量。
首先在AP-1-M的5’端连接上8对GC碱基序列，随后再将AP-1-M和Dox以不同浓度配比进行孵育，孵育2小时后，利用分光光度仪在590nM处比较吸光度高低，最终确定1:10为最佳浓度配比，并以此获得适配体药物偶联体——AP-1-M-Dox。
基于此，本发明的AP-1-M-Dox可以制备用于治疗CD133阳性的肿瘤(如ATC)制剂。
实施例6：AP-1-M-Dox对正常甲状腺细胞和未分化型甲状腺癌细胞的药物毒性作用
如图5所示的适配体AP-1-M的特异性检测图(A：正常甲状腺细胞Nthy-ori3-1；B：未分化型甲状腺癌细胞：FRO)
分别取正常甲状腺细胞株Nthy-ori3-1(NTH)和未分化型甲状腺(ATC)细胞株FRO于96孔板铺板，5000个/孔(含10％胎牛血清的RPMI-1640培养液90ul)；NTH细胞株为对照组A组，FRO细胞株为实验组B组，根据细胞处理方式的不同分为A1组(AP-1-M-Dox处理组)和A2组(Dox处理组)，B1组(AP-1-M-Dox处理组)和B2组(Dox处理组)；随后对于各组不同孔内的细胞加入不同浓度的阿霉素(Dox)或AP-1-M-Dox(10uL/孔)，共9小组，第一组细胞不作处理，作为阴性对照，余8组浓度依次增高2倍，最低浓度约为0.016umol/ml，最高浓度50umol/mL；之后在37℃5％CO2的饱和水汽二氧化碳孵箱中培养3小时，3小时后对所有孔内细胞进行换液处理(含10％胎牛血清的RPMI-1640培养液90ul)；最后在孵箱中培养48小时，最后每孔加10μL CCK，孵箱中培养3h后，用酶标仪测定450nm OD值。
结果说明AP-1-M-Dox对于ATC细胞FRO有良好的细胞毒性作用，但其毒性作用要略弱于Dox，而其对于不表达CD133的正常甲状腺细胞NTH有较弱的细胞毒性作用，其毒性作用明显弱于Dox。
序列表
<110> 浙江省肿瘤医院
<120> 一种靶向结合CD133蛋白的核酸适配体及其筛选方法和用途
<130> CN-CN-2018-0675-1
<141> 2018-12-06
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213> Artificial Sequence
<400> 1
taccagccgt ttccccggag ggtcacccct gacgcattcg gttgac 46
Claims (5)
1.一种靶向结合CD133蛋白的核酸适配体，其特征在于，所述靶向结合CD133蛋白的核酸适配体为单链DNA核酸适配体，所述靶向结合CD133蛋白的核酸适配体序列如序列表中SEQ ID NO.1所示。
2.根据权利要求1所述的一种靶向结合CD133蛋白的核酸适配体，其特征在于，所述靶向结合CD133蛋白的核酸适配体序列5’端还连接有8对GC碱基序列。
3.根据权利要求1所述的靶向结合CD133蛋白的核酸适配体和细胞毒性药物的偶联物在制备治疗未分化型甲状腺癌的药物中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的靶向结合CD133蛋白的核酸适配体偶联毒性药物通过在5’端连接8对GC碱基序列从而结合细胞毒性药物。
5.根据权利要求4所述的靶向结合CD133蛋白的核酸适配体的应用，其特征在于，所述的细胞毒性药物为阿霉素。
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